Among cytomegalovirus (CMV) tegument proteins, phosphoprotein 65 (pp65) has been identified as the important target antigen of the cytotoxic T lymphocyte (CTL) response against the virus. We synthesized seven CMV-pp65-derived peptides carrying an HLA-A24-binding motif, and investigated the ability of these peptides to induce CMV-specific CTL. We identified one nonamer peptide (pp65113-121; VYALPLKML) able to bind HLA-A24 and induce CTL responses in vitro in peripheral blood mononuclear cells (PBMC) from CMV-seropositive individuals. The peptide-specific CTLs generated were capable of recognizing pp65 expressed on CMV-infected fibroblasts as well as pp65113-121 peptide bound to the surface of C1R-A*2402 cells in an HLA-A24-restricted manner. The pp65113-121 peptide thus might be considered a synthetic peptide vaccine in HLA-A24-positive individuals.